Abstract It is increasingly appreciated that the non-coding genome can shape gene expression and, consequently, diseases such as cancer. Beyond a few select examples, however, we know remarkably little about the specific cis- regulatory mechanisms influencing many key oncogenes and tumor suppressors. While various approaches to identify candidate cis-regulatory elements (ccREs) on a genome-wide scale have been successful in nominating large sets putative regulatory regions, many such methods rely on indirect evidence or evidence gleaned outside of the native context of the cell. Further, pairing the regulatory activity of a given ccRE with its gene partners has proven difficult. To address this, our groups created the first technologies for functionally defining the regulatory circuitry of specific genes, CRISPR-Cas9-Based Epigenomic Regulatory Element Screening (CERES). This platform utilizes nuclease-deactivated (d)Cas9 coupled to epigenomic activator (p300) and repressor (KRAB) constructs. These constructs are paired with lentiviral short guide RNA (sgRNA) libraries targeting areas of accessible chromatin surrounding a gene of interest. Thus, we are able to map the effect of both activation and repression of candidate loci on the expression of a target gene, and thereby define the regulatory elements for that gene. Powered by this unique in-house technological approach, we are now interested in defining the endogenous mechanisms controlling expression of important cancer genes. One of the most frequently altered tumor suppressor genes (TSGs) in human cancers is phosphatase and tensin homolog (PTEN), which encodes a lipid and protein phosphatase that negatively regulates the PI3K-AKT pathway, among others. PTEN is mutated or deleted at the genetic level in many tumors. However, a large fraction of patients exhibit loss of PTEN expression without these associated genetic alterations, suggesting a potential role for non-coding alterations controlling PTEN expression. Separately, it is also known that overexpression of PTEN is sufficient to inhibit cancer cell proliferation, drive apoptosis, and stimulate immune surveillance. Therefore, techniques which target endogenous PTEN for overexpression, for instance through the manipulation of its cis-acting regulatory elements (cREs), could represent a promising therapeutic strategy. In this proposal, we will systematically define the key cREs controlling PTEN expression. In so doing, we will provide a foundation for understanding the role of non-coding mutations as drivers of PTEN loss in human tumors and germline tumor syndromes. Further, this work will establish a foundation for identifying the transcription factors and signaling pathways that regulate PTEN expression through these critical cREs, work which could enable the eventual design of therapeutics that target its genetic regulation.